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The Journal of Immunology, 2004, 172: 6578-6586.
Copyright © 2004 by The American Association of Immunologists

Mechanisms of V{delta}1 {gamma}{delta} T Cell Activation by Microbial Components1

Hiranmoy Das*, Masahiko Sugita{dagger} and Michael B. Brenner2,*

* Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and {dagger} Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There are two major subsets of {gamma}{delta} T cell in humans. V{gamma}2V{delta}2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases. Ags identified for the V{delta}2 T cells are nonpeptide phosphate, amine, and aminobisphosphonate compounds. In contrast, V{delta}1-encoded TCRs account for the vast majority of {gamma}{delta} T cells in tissues such as intestine and spleen. Some of these T cells recognize CD1c and MHC class I-related chain B molecules. These T cells are cytotoxic and use both perforin- and Fas-mediated cytotoxicity. A fundamental question is how these {gamma}{delta} T cells are activated during microbial exposure to carry out effector functions. In this study, we provide evidence for a mechanism by which V{delta}1 {gamma}{delta} T cells are activated by inflammatory cytokines in the context of the V{delta}1 TCR. Dendritic cells are necessary as accessory cells for microbial Ag-mediated V{delta}1 {gamma}{delta} T cell activation. Cytokine (IL-12), adhesion (LFA3/CD2, LFA1/ICAM1) and costimulatory (MHC class I-related chain B molecule/NK-activating receptor G2D) molecules play a significant role along with V{delta}1 TCR in this activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Of two major subsets of {gamma}{delta} TCRs in humans, V{gamma}2V{delta}2 T cells predominate in the circulation and significantly expand in vivo during a variety of infectious diseases (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Such expansions can be recapitulated in vitro using extracts from the organisms causing many of these diseases (11, 12, 13). The microbial Ags responsible include negatively charged alkyl phosphate Ags, derived from Mycobacteria (11, 13, 14, 15, 16), and positively charged alkylamine Ags (17). The alkylphosphate and alkylamine Ags are important products of microbes as well as self-Ags. The mechanism by which these Ags are presented is not known, but it does not involve MHC class I, MHC class II, or CD1 molecules (15, 18, 19). We have suggested they may be recognized much as haptens are recognized by either Igs or TCRs (15, 20). Namely, their requirement for an Ag-presenting element is unclear, but their recognition is critically dependent on the CDR3 sequence of the {gamma}{delta} TCR (20, 21). Recently, aminobisphosphonate compounds also were shown to be recognized by circulating V{gamma}2V{delta}2+ T cells via their TCRs (22, 23, 24, 25).

In contrast to V{gamma}2V{delta}2+ T cells that are the major circulating pool of {gamma}{delta} T cells, human {gamma}{delta} T cells bearing V{delta}1-encoded TCRs account for the vast majority of {gamma}{delta} T cells in tissues such as intestine and spleen (26). Recently, selected examples of {gamma}{delta} T cells in this subset were found to recognize the MHC-encoded proteins, MHC class I-related chain A molecule (MICA)3 and MICB (27, 28). Recognition was through the activating NK-activating receptor G2D (NKG2D) C-type lectin (29) and also by the {gamma}{delta} TCR (28). MICA and MICB class I molecules identify stressed cells and also have a restricted pattern of expression, primarily in the intestine. MICA and MICB probably do not present peptides (30); instead, they may function as important targets for V{delta}1+ {gamma}{delta} T cell recognition of stressed cells (27).

The majority of the V{delta}1 {gamma}{delta} T cells are double negative (CD4 and CD8) (31) and cytolytic in nature, using both perforin- and Fas-mediated cytotoxicities (32). These V{delta}1 subsets also produce granulysin, an important antimicrobial protein (32). Upon activation, these subsets of {gamma}{delta} T cells secrete Th1-type cytokines (32). V{delta}1 {gamma}{delta} T cells are expanded in the peripheral blood during HIV (10, 33, 34) and malarial infection (35).

In addition, some V{delta}1+ {gamma}{delta}T cells recognize nonpolymorphic MHC-related CD1c molecules mediated specifically by their TCR (32). This recognition is not dependent on the presence of foreign lipid or glycolipid Ags and probably involves self-lipid molecules presented by CD1c (32). These CD1-specific, V{delta}1-expressing T cells may interact with dendritic cells and B cells as the major cell populations that express CD1c.

Although much has been learned about the diversity, development, and homing of {gamma}{delta} T cells, a fundamental question is how they are activated to carry out effector functions. In this study, we provide evidence that V{delta}1 {gamma}{delta} T cells are activated and expanded in response to lipid extracts of Gram-negative bacteria in the presence of monocyte-derived dendritic cells. We found that inflammatory cytokines and costimulatory molecules activated by microbial products cooperate to activate V{delta}1-bearing {gamma}{delta} T cells. This may provide a major mechanism for V{delta}1 {gamma}{delta} T cell expansion during bacterial infection in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monoclonal Abs

The following mAbs were used for flow cytometry and blocking experiments: P3 (IgG1 control), IgG2a and IgG2b (BD PharMingen, San Diego, CA), SPV-T3b (anti-CD3), OKT3 (anti-CD3) anti-TCR-{delta}1 (pan anti-C{delta}), {delta}TCS1 (anti-V{delta}1/J{delta}1), Ti{gamma}A (anti-V{gamma}2), 9.3 (anti-CD28), OKT4 (anti-CD4; American Type Culture Collection, Manassas, VA), OKT8 (anti-CD8{alpha}; American Type Culture Collection), DX1 (anti-NKR-P1A; provided by Dr. L. Lanier, DNAX, Palo Alto, CA), BMAO31 (pan anti-TCR-{alpha}{beta}; provided by Dr. R. G. Kurrle, Boehringwerke, Marburg, Germany), 7C6 (anti-CD1c), F10/21A3 (anti-CD1c), BCD1b3.2 (anti-CD1b), 10H3.9.3 (anti-CD1a), W6/32 (anti-MHC class I; American Type Culture Collection), L243 (anti-HLA-DR; American Type Culture Collection), NS4.1 (IgM control; American Type Culture Collection), 4A11 (anti-V{gamma}1.4), anti-IL-12 (BD PharMingen), anti-CD2 (TS2/18), anti-LFA1 (BD PharMingen), anti-LFA3 (TS2/9), anti-{alpha}V{beta}3 (69.6.5), anti-NKG2D (1D11, 5C6), and anti-MICA (6D4) both mAbs were provided by Dr. T. Spies (Fred Hutchinson Cancer Research Center, Seattle, WA). The specificity of some mAbs was previously reviewed (36). Tissue culture medium RPMI 1640 and supplements were purchased from Life Technologies (Grand Island, NY). FBS was obtained from HyClone Laboratories (Logan, UT).

Immunofluorescence analysis

Cells were incubated with mouse mAbs on ice for 30 min, washed, and stained with FITC-conjugated F(ab')2 goat anti-mouse Ig (Tago Scientific, Burlingame, CA) for an additional 30 min on ice in a two-step method or were used directly conjugated with FITC or PE mAbs in one-step staining. After washing, the cells were resuspended in propidium iodide and analyzed by flow cytometry (FACSort; BD Biosciences, Mountain View, CA). Results were expressed as the percentage of positive cells compared with negative cells stained with isotype-matched control mAbs.

T cell lines and clones

Lymphocytes were isolated from fresh peripheral blood of random healthy donor leukopacks as a byproduct of plateletpheresis at Dana-Farber Cancer Institute (Boston, MA) by Ficoll-Hypaque centrifugation. {gamma}{delta} T cells were enriched by staining with anti-TCR-{alpha}{beta} mAb and BMAO31 and CD4+ T cells with OKT4, respectively, followed by depletion of {alpha}{beta} T cells and CD4+ T cells with magnetic beads coated with goat anti-mouse IgG (Dynal Biotech, Great Neck, NY). T cell lines were established from two separate donors by culturing 1 x 106 freshly isolated {gamma}{delta} T cells in 1-ml culture wells with 1 x 106 irradiated (5000 rad) autologous monocyte-derived dendritic cells as accessory cells and the organic phase of a chloroform/methanol (2/1) extract of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Mycobacterium tuberculosis prepared as previously described (37, 38) at a 40 µg/ml final dilution. After 2 wk of culture, viable cells were recovered, and the residual {alpha}{beta} T cells and CD4+ T cells were depleted with the above-mentioned protocol using magnetic beads (Dynal Biotech). The resulting population was restimulated with autologous dendritic cells and total lipid extract of respective microbes, and supplemented with rIL-2 (1 nM; Ajinomoto, New York, NY). The V{delta}1 {gamma}{delta} T cell line SL1 was subjected to positive selection for V{delta}1 T cells using FITC-conjugated, V{delta}1-specific mAb {delta}TCS1 and using magnetic beads against FITC in a MACS column separation technique (Miltenyi Biotec, Auburn, CA). These homogeneous T cell lines were maintained by restimulation every 2 wk with irradiated heterologous dendritic cells and rIL-2. T cell clones were derived from early lines by limiting dilution culture using PHA stimulation. T cell cloning was performed as previously described (39). In brief, T cells were seeded at 1 and 0.5 cells/well in 96-well, round-bottom plates in a volume of 0.2 ml, with 5 x 104 irradiated (4000 rad) heterologous PBMCs and 5 x 104 irradiated (5000 rad) EBV-transformed B lymphoblastoid cells as feeders in RPMI 1640 medium supplemented with PHA (1/4000 final dilution; Difco, Detroit, MI) and IL-2 (1 nM).

APC lines

Monocyte-derived dendritic cells were generated from human blood monocytes that were isolated from the byproducts of platelet pheresis and induced to differentiate and express CD1a, CD1b, CD1c, and MHC class II by incubation with GM-CSF and IL-4 as previously described (40). The mutant cell lines T2 were transfected with the expression vector pSR{alpha}-Neo into which cDNAs encoding CD1a, CD1b, or CD1c were inserted as described previously (41).

Proliferation assays

T cells (3 x 104) were plated in triplicate in 96-well, flat-bottom plates with either 5 x 104 mitomycin C-treated heterologous dendritic cells or T2 cells transfected with CD1a, CD1b, or CD1c as APCs. In the mAb blocking experiments, mAbs were added as ascites (final initial dilution of 1/200 and log dilutions) or purified mAbs (final initial dilution of 20 µg/ml and log dilutions) to the T cells or monocyte-derived dendritic cells, incubated for 45 min at 37°C, and washed, then APC or T cells and bacterial products were added. Cultures were incubated at 37°C, pulsed with 1 µCi of [3H]thymidine (6.7 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) on day 3, and harvested 6 h later using a Tomtec harvester (Hamden, CT). The filter papers were counted on a Betaplate scintillation counter (Wallac, Gaithersburg, MD). Results were expressed as cpm ± SEM.

Cytokine assay

T cells (3 x 104) were cultured with 5 x 104 heterologous monocyte-derived dendritic cells as APC. PHA (1/4000) was added as a positive control. Supernatants were harvested after 24 and 48 h of culture. Cytokine release was determined for IFN-{gamma} by sandwich ELISA (42) using Ab pairs purchased from BD PharMingen. Results were expressed as nanograms per milliliter ± SEM.

Bacterial culture

Gram-negative bacteria, E. coli and S. typhimurium (SL3261), and Gram-positive bacteria, S. aureus and M. tuberculosis (H37Ra), obtained from American Type Culture Collection, were grown in 7H9 broth (Difco) containing 10% albumin dextrose catalase supplement (Difco), 2% glycerol, and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO) typical of log phase growth up to an OD600 of 1.0.

Bacterial extracts

Bacterial extractions were performed by following previously described protocols (37, 38). In brief, bacteria were washed first with PBS, then with deionized water, lyophilized, and extracted for 2 h in chloroform/methanol (2/1) to give total extractable lipid (C/M). Folch extractions of the C/M were performed for 2 h using chloroform/methanol/water (2/1/1), and phases were separated after centrifugation. Each phase or fractions were evaporated or lyophilized and resuspended in appropriate solvents. Open silica columns were 2 x 20 cm, and C/M extracts were eluted sequentially by nonpolar hydrophobic solvent, followed by polar hydrophilic solvents, including chloroform, acetone, and methanol, and finally with water.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To identify microbial components that might be capable of activating {gamma}{delta} T cells but were distinct from polyprenylphosphates previously described, we took the approach of solubilizing microbes in organic solvents to obtain candidate lipid Ags. This approach could also extract lipid-containing molecules that stimulate cells through CD1 lipid-binding, Ag-presenting molecules or through Toll-like receptors.

Generation of microbial product-stimulated {gamma}{delta} T cell lines

To focus on the reactivity of {gamma}{delta} T cells, PBMCs were depleted of CD4+ and {alpha}{beta}+ T cells by mAb staining and magnetic bead separation. When analyzing the phenotypic distribution of V{delta}1 vs V{delta}2 T cells in the total PBMC, we consistently found that V{delta}2 T cells were 5–7 times higher than V{delta}1 T cells. For the experiment in Fig. 1A, the initial V{delta}2 T cells were 3.1% and V{delta}1 T cells were 0.5% of the total PBMC. The {gamma}{delta} T cell-enriched PBMCs then were cultured in vitro with a total lipid extract using a C/M extraction procedure of a Gram-negative bacteria (E. coli or S. typhimurium), Gram-positive bacteria (S. aureus), or mycobacteria (M. tuberculosis) in the presence of autologous monocyte-derived dendritic cells (see Materials and Methods). This stimulation resulted in marked proliferation of leukocytes, with the striking expansion of V{delta}1 {gamma}{delta} T cells with in 2 wk, as analyzed by flow cytometry. For example, Gram-negative bacteria E. coli stimulated a culture containing 24% (a 48-fold expansion) V{delta}1+ T cells, and S. typhimurium total lipid extracts stimulated 32% (a 64-fold expansion) V{delta}1 {gamma}{delta} T cells. In contrast, S. aureus and mycobacteria total lipids stimulated only 8 or 4% V{delta}1+ T cells, respectively (Fig. 1A). Without Ag stimulation, we recovered 7% V{delta}1 T cells, which was considered a background level in this {alpha}{beta} T cell-depleted culture system. Trends were also similar in five other experiments. Note that many non-T cells (probably NK cells (CD3–, CD56+; data not shown) were also expanded using this culture system (Fig. 1A; anti-CD3 staining). Short term T cell lines were then derived from an S. typhimurium extract stimulated line following repeated magnetic bead depletion and stimulation with similar lipid extracts and were examined for their reactivity against microbial C/M extracts of E. coli, S. typhimurium, S. aureus, or M. tuberculosis. Characterization of a representative short term line raised against the S. typhimurium extract revealed the expansion of CD3+ T cells (83% of the line), of which 99% were V{delta}1 (Fig. 1B, upper panel). Seventeen percent of the cells of the line were probably NK cells (CD3–) that also appeared to be expanded by this method. Using this in vitro coculture system, {gamma}{delta} T cells proliferated against Gram-negative bacterial C/M extracts, but not against Gram-positive bacteria or mycobacterial extracts (Fig. 1B, lower panel). E. coli extracts were found to be the most potent.



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  		FIGURE 1. Gram-negative microbial total lipid extracts mediate V{delta}1 {gamma}{delta} T cell proliferation. A, TCR {alpha}{beta} and CD4+ T cell-depleted PBMCs were cultured with autologous monocyte-derived dendritic cells with 40 µg/ml total lipid extract of E. coli, S. typhimurium, S. aureus, or M. tuberculosis, and after 2 wk cells were analyzed by flow cytometry. {blacksquare}, Specific mAb of CD3, pan {gamma}{delta}, or V{delta}1; {square}, isotype control mAb. The two upper panels show an expansion of V{delta}1 {gamma}{delta} T cells with E. coli (24%) and S. typhimurium (32%) total lipid extracts, respectively. The two lower panels with total lipid extract from S. aureus (8%) and M. tuberculosis (4%) did not show expansion of V{delta}1 T cells. Without bacterial lipid, we recovered 7% of the V{delta}1 T cells; they did not expand and were enriched by depletion procedure. B, With repeated depletion and restimulation with S. typhimurium total lipids, we generated a V{delta}1 T cell line. Two weeks after the last stimulation, cells were evaluated by flow cytometric analysis. Eighty three percent of cells were CD3+, and all CD3+cells were {gamma}{delta} T cells with specific TCR V{delta}1 ({blacksquare}). {square}, Isotype controls (upper panel). The short term V{delta}1 {gamma}{delta} T cell line, SL1, was incubated with monocyte-derived dendritic cells alone or in the presence of total lipid (C/M) extracts of various Gram-negative, Gram-positive, and mycobacterium with increasing amounts for a T cell proliferation assay (lower panel). [3H]Tdt incorporation was measured as a parameter for T cell activation. Dose-dependent proliferation was noted with Gram-negative bacteria, not with Gram-positive or mycobacterial total lipid extracts. Results are representative of at least three independent experiments.

 
Generation of microbial product-reactive {gamma}{delta} T cell clones

To further characterize the basis for reactivity against microbial products, we obtained a panel of clonal populations of V{delta}1 {gamma}{delta} T cells by limiting dilution cloning from a line generated against S. typhimurium. Four independent clones were examined by flow cytometric analysis and revealed expression of the V{delta}1 chain paired with V{gamma}1 chains (Fig. 2A). To evaluate reactivity against microbial molecules, C/M extracts of E. coli, S. typhimurium, S. aureus, or M. tuberculosis were added, and T cell proliferation was determined by [3H]Tdt incorporation. Reactivity against Gram-negative bacterial extracts was highly pronounced in a dose-dependent fashion in all V{delta}1 clones tested (Fig. 2B). For instance, proliferation of clone SL1.12 was 44,544 cpm with 10 µg/ml E. coli extract and 41,423 cpm with 33 µg/ml S. typhimurium extract, whereas using 100 µg/ml Gram-positive (S. aureus) bacterial extract, proliferation was only 1,569 cpm, and using 33 mg/ml M. tuberculosis extract, 3658 cpm were observed. Without bacterial extracts, very low proliferation of only 503 cpm was noted (Fig. 2B). A similar pattern was observed with clone SL1.1. The proliferation of clone SL1.4 was 2,311 cpm without bacterial extracts, 11,087 cpm with 33 µg/ml M. tuberculosis extract, and 11,199 cpm with 100 µg/ml S. aureus extract. Proliferation with Gram-negative bacterial extracts was much higher: 28,546 cpm with 33 µg/ml E. coli extract and 44,831 cpm with 33 µg/ml S. typhimurium extract. Similarly, clone SL1.11 incorporated 16,451 cpm with 33 µg/ml M. tuberculosis extract and 12,518 cpm with 33 µg/ml S. aureus extract compared with the no-Ag control (5,006 cpm; Fig. 2B). Much higher proliferation was observed with Gram-negative bacterial extracts, such as 52,483 cpm with 10 µg/ml E. coli extract and 60,565 cpm with 33 µg/ml S. typhimurium extract, respectively. These responses to C/M extracts of Gram-negative bacteria were not observed for either V{gamma}2V{delta}2 T clones (Fig. 2C, upper panel) or {alpha}{beta} T cell clones (Fig. 2C, lower panel). However, as expected, the V{delta}2 T cell clone was reactive against specific bisphosphonate Ag risedronate in a dose-dependent fashion, and the {alpha}{beta} T cell clone proliferated with nonspecific stimulus PHA (Fig. 2C).



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  		FIGURE 2. V{delta}1 T cell clones display reactivity against Gram-negative microbial lipid extracts. A, Flow cytometric analysis of representative V{delta}1 {gamma}{delta} T cell clones SL1.12, SL1.4, SL1.1, and SL1.11, which were generated by limiting dilution cloning techniques from an S. typhimurium total lipid-derived, early V{delta}1 line. {square}, Isotype controls; {blacksquare}, respective markers as indicated. B, Representative V{delta}1-specific T cell clones were incubated with monocyte-derived dendritic cells in the presence or the absence of total lipid (C/M) extracts from various Gram-negative, Gram-positive, and mycobacteria in a T cell proliferation assay. T cell proliferation against Gram-negative bacterial extracts was highly pronounced with all clones tested. Small amounts of proliferation were also observed with total lipid extracts of S. aureus and M. tuberculosis with SL1.4 and Sl1.11 clones. Insignificant or no proliferation was found without microbial lipids. Results are representative a panel of clones tested in several independent experiments. C, Proliferation with Gram-negative bacterial extracts was not found with using the V{delta}2 T cell clone (upper panel) or the {alpha}{beta} T cell clone (lower panel) with similar coculture procedures. A dose-dependent proliferation of the V{delta}2 T cell clone was observed with risedronate, a TCR-specific Ag of V{delta}2 T cells. whereas PHA-mediated proliferation was observed with the {alpha}{beta} T cell clone.

 
Known Ag-presenting molecules are not required for activation of V{delta}1 clones

An earlier report from this laboratory indicated that some V{delta}1 T cells are CD1c restricted and self-Ag reactive (32). To evaluate the possible role of Ag-presenting molecules in the presentation of microbial products of Gram-negative bacteria to {gamma}{delta} T cells, two series of experiments were performed. First, V{delta}1 T cell proliferation after coculture with C/M extracts of E. coli in presence of dendritic cells was performed, after attempted blockade using mAb against CD1a, -b, -c, and -d; MHC-I; or MHC-II. No significant blocking of proliferation was noted with mAb against any of these known Ag-presenting molecules (Fig. 3A).



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  		FIGURE 3. Known Ag-presenting molecules are not involved, but dendritic cells are required in V{delta}1 {gamma}{delta} T cell activation by microbial lipids. A, The V{delta}1 T cell clone SL1.3 was incubated with monocyte-derived dendritic cells and C/M extracts from E. coli (33 µg/ml) with or without added purified mAbs (20 µg/ml) against CD1a, CD1b, CD1c, CD1d, MHC-I, MHC-II, or isotype control mAb. [3H]Tdt incorporation was measured as a parameter for cell proliferation. No significant blocking was observed with any of the mAbs against known Ag-presenting molecules. Minor blocking noted in this particular experiment with mAbs against MHC classes I and II was not observed in any other experiments. Results are representative of experiments using several independent clones tested in different experiments. B, EBV-transformed B cells (DG.EBV) failed to present microbial lipid extracts to mediate proliferation of V{delta}1 T cell clone SL1.3, whereas monocyte-derived dendritic cells were capable of inducing proliferation. C, T1 cells express MHC class I and II molecules, whereas T2 mutant cells do not. T2 cells transfected with TAP express class I molecules, and T2 cells transfected with HLA-DM and HLA-DR3 express class II molecules. In addition, T2 cells were transfected to express CD1a, CD1b, or CD1c. V{delta}1 T cell proliferation was measured using E. coli total lipid extracts in a dose-dependent manner. Monocyte-derived dendritic cells induced V{delta}1 T cell proliferation, whereas none of the T2 mutant cells was able to function as APC. This is a representative of four independent clones tested.

 
In the second experimental series, either EBV-transformed B cells (Fig. 3B) or a mutant cell line, T2, that has deleted the peptide transporter system impairing MHC class I loading and also lacks expression of MHC class I and II, and any CD1 isoforms, was transfected with CD1a, -b, -c, and -d; MHC-I; or MHC-II molecules and used as an APC during V{delta}1 T cell proliferation assays in the presence of E. coli C/M extracts (Fig. 3C). Expression of the CD1 isoforms, TAP (MHC-I), or MHC class II molecules did not enable T2 cells to stimulate V{delta}1 T cells in the presence of potent microbial extracts (Fig. 3C). Only monocyte-derived dendritic cells were found to activate V{delta}1 T cells (Fig. 3, B and C). These experiments suggested that none of the known major Ag-presenting molecule groups conferred the ability to activate V{delta}1 T cells by microbial organic extracts, but a critical requirement for monocyte-derived dendritic cells was apparent.

Polar lipid nature of the active microbial products

To identify the nature of the microbial molecule responsible for V{delta}1 T cell activation, we separated the components of the total organic extracts. Two different approaches were taken to further purify E. coli C/M extracts. First a Folch extraction of the E. coli extract was made to generate three separate phases (organic, aqueous, and interphase). Analysis of these phases revealed the major reactivity to be in the interphase (not shown).

Second, the E. coli C/M extract was loaded onto an open silica column and eluted sequentially with solvents of increasing polarity (chloroform, acetone, methanol, and water). These elutes were collected, lyophilized, resuspended in the medium at appropriate concentrations, and used to determine V{delta}1 T cell responses for either T cell proliferation (Fig. 4A) or cytokine production (IFN-{gamma} secretion; Fig. 4B). The water-eluted fraction from the silica column contained the major bioactivity (Fig. 4, A and B), which is partially purified, as the major portion of lipids eluted from the silica column with methanol and did not show the bioactivity. This reactivity is 0.5 log greater than that of the total lipid extract reactivity measured by T cell proliferation or IFN-{gamma} production. Nonpolar solvent-eluted molecules did not show biological activity. As the molecules were first extracted from E. coli using organic solvents, the subsequent elution in polar solvents from silica columns suggested that they might be polar lipids.



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  		FIGURE 4. Isolation of biologically active molecules from total lipid extracts of E. coli. A, Total lipid extracts were fractionated using an open silica column by eluting with solvents of increasing polarity, such as chloroform, acetone, methanol, and water. Eluted molecules were lyophilized, resuspended in equivalent quantities by weight in half-log dilutions (highest concentration was 33 µg/ml), and used as stimulants in V{delta}1 T cell proliferation assays. Monocyte-derived dendritic cells were used as accessory cells in all experiments. A dose-dependent proliferation was observed with the crude C/M extract and water-eluted fraction. The water-eluted fraction was more potent then the total lipid extracts. Representative results of one of three experiments are shown. Each experiment was performed in triplicate, and the mean value was plotted. B, T cell culture supernatants of the duplicate experiment of A were collected after 24 h and used to evaluate IFN-{gamma} secretion in supernatants with a sandwich ELISA technique (BD PharMingen). IFN-{gamma} secretion correlated with the proliferation observed in the experiment (A). C, In a separate experiment, pure LPS isolated from of E. coli was compared with the total lipid extract from E. coli in a V{delta}1 T cell proliferation assay. The highest concentrations for total lipid extracts and LPS were 33 and 1 µg/ml, respectively. Monocyte-derived dendritic cells were used as accessory cells. The results shown are representative of three independent clones tested.

 
One of the prominent polar lipids in the E. coli cell wall is LPS. Limulus amebocyte lysate assays revealed that silica column fractions with the most bioactivity also were positive in the Limulus assays. To determine whether the V{delta}1 T cells were responding to LPS in the presence of monocyte-derived dendritic cells, we used pure LPS from E. coli (Sigma-Aldrich) in T cell proliferation assays. We found that V{delta}1 T cells proliferated in the presence of pure LPS-pulsed, monocyte-derived dendritic cells (Fig. 4C).

Possible role for V{delta}1 TCR in recognition of microbial products and dendritic cells

To assess the potential involvement of {gamma}{delta} TCR in the response to microbial lipid-mediated activation, we performed mAb blocking against the {gamma}{delta} TCR. V{delta}1-specific mAb ({delta}TCS1) blocked T cell proliferation 75% in a dose-dependent manner (Fig. 5), whereas isotype control did not, implicating an involvement of TCR. However, efforts to confer reactivity of T cell clones by transfection of their TCRs into TCR-deficient Jurkat cells were not successful (data not shown). This failure might be due to the inability of the cells (Jurkat) to up-regulate non TCR surface molecules necessary for reactivity, such as costimulatory molecules involved in T cell APC interactions.



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  		FIGURE 5. Involvement of V{delta}1 TCR in microbial lipid-induced T cell activation. SL1.13 V{delta}1 T cells were incubated with monocyte-derived dendritic cells and the water-eluted fraction of total lipids from E. coli (33 µg/ml) in the presence or the absence of mAbs ({delta}TCS1 ascites; highest concentration was 1/200) or isotype control mAb. [3H]Tdt incorporation was measured as a parameter for cell proliferation. T cells were incubated for 45 min with various concentrations of V{delta}1-specific mAb or isotype control mAb at room temperature, washed three times with media, then stimulated with an equal concentration of water elute (33 µg/ml) in the presence of monocyte-derived dendritic cells. Dose-dependent blocking was observed with V{delta}1-specific mAb, but not with the isotype controls. Results are representative of three independent clones tested in different experiments.

 
Microbial products induce secretory products from APC that are important in T cell activation

Together the above findings led us to consider that the microbial products might be critical in activating the monocyte-derived dendritic cells in ways that were responsible for activating the V{delta}1 T cell clones. Heterodimeric IL-12 is a dominant factor produced by dendritic cells (43). Earlier studies have emphasized the contributions made by both innate microbial signals and T cell help to obtain maximal heterodimeric IL-12 production by dendritic cells (44, 45). As IL-12 would probably be produced in our coculture system and is known to provide a signal that can stimulate T cell proliferation (46), we examined its role in V{delta}1 T cell proliferation to microbial stimulation. Using either a water-eluted fraction from silica column of total lipid extracts of E. coli origin or pure LPS from a commercial source, inhibition of proliferation of 72% was noted using anti-IL-12 mAb (Fig. 6A).



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  		FIGURE 6. IL-12 plays a significant role in activation of V{delta}1 T cells in the presence of monocyte-derived dendritic cells. A, V{delta}1 T cells were cultured with monocyte-derived dendritic cells, microbial extract (water elute of silica column, 100 µg/ml), and anti-IL-12 mAb or control mAb. T cell proliferation was measured by [3H]Tdt incorporation. Dose-dependent blocking with anti-IL-12 mAb was observed, and no blocking was found with the isotype-matched control mAb. B, V{delta}1 T cells were cultured in the presence or the absence of monocyte-derived dendritic cells with or without rIL-12 (2 ng/ml) in a T cell proliferation assay. No microbial lipid extracts were present. V{delta}1 T cells proliferated with only rIL-12 in the presence of dendritic cells. C, For comparison, V{delta}2 T cells were also cultured in the presence or the absence of monocyte-derived dendritic cells with or without rIL-12 (2 ng/ml) in a similar T cell proliferation assay. V{delta}2 T cell proliferation was not significant with rIL-12 in the presence or the absence of dendritic cells. At the same time, using this V{delta}2 clone, T cell proliferation against the V{delta}2-specific Ag risedronate (19,318 cpm) was noted (not shown).

 
To confirm the role of IL-12 in activation, V{delta}1 T cell clones were cultured in the presence or the absence of monocyte-derived dendritic cells without microbial products, but with the addition of rIL-12, which potently mediated proliferation of the V{delta}1 T cell clone, but only in presence of dendritic cells. In the absence of dendritic cells, IL-12 failed to mediate any proliferation (Fig. 6B). IL-12-driven proliferation was not typically observed with V{delta}2 T cell clones in either the presence or the absence of monocyte-derived dendritic cells (Fig. 6C). The requirement for dendritic cells in this culture system with exogenously added rIL-12 suggested that besides cytokines, some other molecules present in dendritic cells might be involved in the V{delta}1 T cell activation.

Involvement of costimulatory molecules in microbial product-mediated V{delta}1 T cell activation

As IL-12 did not mediate T cell activation without dendritic cells, we speculated that costimulatory molecules up-regulated on monocyte-derived dendritic cells by microbial products might be critical for V{delta}1 T cells proliferation. Thus, we tested the roles of several adhesion and costimulatory molecules in V{delta}1 T cell proliferation assays. We found a significant dose-dependent blocking by mAb against LFA3 of 68% (Fig. 7A). Blocking with mAb against CD2 (the counter-receptor expressed in T cells for LFA3) in the proliferation assay reached a maximum of 76% in a dose-dependent fashion (Fig. 7B). mAb against another adhesion molecule, LFA1, which is present in APC and T cells, partially inhibited proliferation (54%; Fig. 7D). In contrast, mAb against {alpha}V{beta}3, another integrin expressed on these T cells, did not show any blocking of proliferation (Fig. 7C). Other important, recently identified costimulatory molecules found to be expressed in the intestinal epithelial cells are MICA and MICB (47). mAb Against MICA showed partial inhibition of activation by as much as 52% (Fig. 7E). The counterligand for MICA, which is expressed in most human {gamma}{delta} T cells, is an activating molecule, NKG2D (29, 48). mAb against NKG2D also inhibited proliferation partially (40%; Fig. 7F).



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  		FIGURE 7. Adhesion and costimulation molecules are critical for V{delta}1 T cell activation with Gram-negative microbial products. V{delta}1 T cells or monocyte-derived dendritic cells were incubated for 45 min with various concentrations of mAbs against LFA-3 (TS2/9; A), CD2 (TS2/18; B), {alpha}V{beta}3 (69.6.5; C), LFA-1 (BD PharMingen; D), NKG2D (5C6; E), or MICA (6D4; F) and washed three times with T cell medium before culture with monocyte-derived dendritic cells or T cells in the presence of water-eluted E. coli extract (33 µg/ml) in a T cell proliferation assay. {blacktriangleup}, Isotype-matched control mAb; {blacksquare}, blocking mAb. Dose-dependent blocking was observed with mAbs against LFA-3, CD2, LFA-1, NKG2D, and MICA. However, we did not detect any blocking with mAb against {alpha}V{beta}3 and isotype control Abs. Anti-MICA and anti-NKG2D mAbs were ascites, and the highest concentrations were used at 1/200 dilutions; others are purified mAbs used at the concentrations stated. These experiments were repeated three times with three different clones, and similar patterns were observed; one representative is shown.

 
Thus, molecular interactions between activating receptors on V{delta}1 T cells (such as NKG2D, LFA-1, and CD2) and their counter-receptors on dendritic cells (such as MICA/B and LFA-3) are of critical importance in the activation of T cells. Based on these in vitro studies we propose that the mechanism involved in activation of the tissue pool of V{delta}1 {gamma}{delta} T cells found in vivo by microbial stimulation may involve cytokine secretion and costimulation mediated by dendritic cells (Fig. 8). The data for V{delta}1 expression and anti-TCR {gamma}{delta} blocking presented suggest that TCR {gamma}{delta} is required, although the specific Ag stimulating {gamma}{delta} TCR are not known. Nevertheless, in the presence of microbial activation of dendritic cells, the production of IL-12 and the cell-to-cell interaction involving adhesion and costimulatory molecules appear to selectively activate V{delta}1 and not V{delta}2 {gamma}{delta} T cells (Fig. 8).



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  		FIGURE 8. Possible model of molecular interactions in V{delta}1 T cell activation by Gram-negative microbial products. Monocyte-derived dendritic cells may be activated by exposure to Gram-negative bacterial lipid components, such as LPS, which activates dendritic cells through Toll-like receptors (TLRs) and results in secretion of IL-12. Microbial Ags or self-Ags expressed by dendritic cells might also activate V{delta}1 TCR. During coculture, interaction takes place between various adhesion and costimulatory molecules present in both T cells (CD2, ICAM-1, and NKG2D) and dendritic cells (LFA-3, LFA-1, and MICA). Together these signals stimulate proliferation, cytokine secretion, cytotoxic lysis, granulysin secretion, and other {gamma}{delta} T cell effector functions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence suggest that {gamma}{delta} T cells play an important role in host defense and immunoregulation. Recent insights have been gained by the discovery of recognition of unique nonpeptide Ags, such as isopentenyl pyrophosphate (49), and alkylamine Ags, such as iso-butylamine (17), that are secreted by bacteria and are recognized in a V{gamma}2V{delta}2 TCR-dependent manner (20). These Ags are most likely responsible for triggering V{gamma}2V{delta}2 T cells to mediate antibacterial effector functions such as proliferation (12), secretion of TNF-{alpha} and IFN-{gamma} (50), secretion of chemokines (51), mobilization of granulysin (32), and cytotoxicity (15). Further, V{gamma}2V{delta}2 T cells display a memory-type response to these Ags in Calmette-Guérin bacillus-vaccinated macaque monkeys and provide immunity to subsequent challenge with Mycobacterium tuberculosis (52).

In contrast, human V{delta}1 {gamma}{delta} T cells are the majority of {gamma}{delta} T cells in tissues such as spleen and intestine (26), yet the Ags they recognize remain enigmatic. Recent studies have demonstrated that some V{delta}1 {gamma}{delta} T cells can recognize stress-inducible MHC class I-like MICA/B molecules expressed by epithelial cells via specific TCR interactions (28). We have previously demonstrated that other {gamma}{delta} T cells expressing V{delta}1-encoded TCRs recognize CD1c, one member of a family of nonpolymorphic CD1 molecules expressed on the surface of dendritic cells and B cells, that present lipid and glycolipid foreign Ags to T cells (32).

In this study we developed a system in which V{delta}1 {gamma}{delta} T cells could be expanded by microbial products. We demonstrate for the first time that V{delta}1 {gamma}{delta} T cells strikingly respond to total lipid extracts of Gram-negative bacteria, whereas V{delta}2 {gamma}{delta} T cells did not display this response. Reactivity to hydrophobic Gram-negative bacterial extracts was unexpected and was clearly distinct from previously reported reactivity to organic phosphates. Those earlier findings were made with the total sonicates of the M. tuberculosis that selectively expanded the V{delta}2 subset of {gamma}{delta} T cells (15, 16), not V{delta}1 {gamma}{delta} T cells. Although all the bioactive molecules of Gram-negative bacteria that stimulate V{delta}1 {gamma}{delta} T cells have not been determined, the water-eluted fraction of the silica column loaded with E. coli C/M extract contained the major reactivity. As LPS is enriched in this fraction, we tested it directly and confirmed it to be bioactive. Other bioactive molecules may also exist in Gram-negative bacterial organic extracts that can similarly activate V{delta}1 {gamma}{delta} T cells. The activation of V{delta}1 {gamma}{delta} T cells occurred only in the presence of monocyte-derived dendritic cells, not with B cell or tumor cell APCs. Interestingly, none of the known major Ag-presenting molecule groups conferred the ability to activate V{delta}1 T cells by microbial lipid extracts. As dendritic cells served as potent intermediaries of the response, but did not appear to be using the known Ag-presenting molecules, we considered their capacity for costimulation and cytokine production.

In this present study dendritic cells were incubated with bacterial products for 24 h, and after centrifugation, supernatants transferred to T cells failed to mediate proliferation (data not shown). This suggested that T cell and dendritic cell contact was critical to mediate V{delta}1 {gamma}{delta} T cell activation. Further, we found that IL-12 produced by dendritic cells provided a major cytokine signal for driving V{delta}1 {gamma}{delta} T cell proliferation in this system. Although microbial products such as LPS can effectively promote the initial production of IL-12 by dendritic cells, in the absence of additional host-derived signals such as IFN-{gamma}, these dendritic cells rapidly lose the capacity to produce IL-12 upon subsequent CD40 ligation by naive T cells (43, 46). Fundamentally, the mechanism proposed in this study for {gamma}{delta} T cells is similar to that recently characterized in our laboratory, in which microbial TLR agonists stimulate IL-12 production by APCs that then activate NKT cells in a CD1d-TCR dependent manner (53).

Adhesion molecule interactions serve two broad functions: 1) to promote binding of T cells to APC, and 2) to modulate the signaling events that result from Ag recognition. Thus, the ability to block {gamma}{delta} T cell activation by mAb to LFA-1 suggests a critical role for adhesion mediated by this integrin. The interaction of CD2 and LFA-3 also was critical in T cell activation by microbial products. Other recently identified costimulatory molecules, MICA and MICB (47), interact with NKG2D present in {gamma}{delta} T cells and enable signal transduction (29, 48). Human NK cells and V{delta}1 T cells bearing NKG2D receptors can lyse tumor cells bearing MICA and target cells transfected with MICA genes, or M. tuberculosis-infected monocytes or epithelial cells, and this lysis can be blocked using Abs to MICA or NKG2D (27, 29, 39). We found a key role for MICA and NKG2D in the activation of V{gamma}1V{delta}1 T cells by microbial lipid products in conjunction with dendritic cells.

As the capacity of Gram-negative microbial products to stimulate {gamma}{delta} T cells was clearly different for V{delta}1- and V{delta}2-expressing T cells, we considered the role of TCR recognition in this system. We could selectively block the proliferation of V{delta}1 T cell clones using V{delta}1 J{delta}1-specific mAb ({delta}TCS1) in a dose-dependent manner. This suggests a direct role for V{delta}1 TCR in the T cell activation process. However, at present the nature of the Ags recognized by TCR in this system is not known. Together these studies provide the first example of selective expansion of the major tissue pool of {gamma}{delta} T cells (V{delta}1+) by microbial products. We suggest that costimulatory CD2/LFA-3, ICAM-1/LFA-1, NKG2D/MICA, and IL-12 work in concert with V{delta}1 TCR to activate the tissue pool of {gamma}{delta} T cells in response to microbial Ags (Fig. 8).


   Acknowledgments
 
We thank Jack F. Bukowski, M. S. Vincent, and Christopher C. Dascher for their valuable suggestions, and C. Morehouse for technical assistance.


   Footnotes
 
1 This work was supported by a grant from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and Harvard Medical School, One Jimmy Fund Way, Boston, MA 02115. E-mail address: mbrenner{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: MICA, MHC class I-related chain A molecule; C/M, chloroform/methanol; MICB, MHC class I-related chain B molecule; NKG2D, NK-activating receptor G2D. Back

Received for publication January 8, 2004. Accepted for publication March 23, 2004.


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